CN112322743A - Kit for detecting human SEPT9 gene methylation and use method and application thereof - Google Patents
Kit for detecting human SEPT9 gene methylation and use method and application thereof Download PDFInfo
- Publication number
- CN112322743A CN112322743A CN202011414930.3A CN202011414930A CN112322743A CN 112322743 A CN112322743 A CN 112322743A CN 202011414930 A CN202011414930 A CN 202011414930A CN 112322743 A CN112322743 A CN 112322743A
- Authority
- CN
- China
- Prior art keywords
- dna
- probes
- methylation
- kit
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The invention discloses a kit for detecting human SEPT9 gene methylation and a use method and application thereof. The kit comprises specific primers and probes for detecting or measuring the methylation state or level of SEPT9 gene in the test sample DNA. The kit can obviously improve the positive detection rate and specificity (real negative rate) of early liver cancer, can improve the detection sensitivity of the biomarker to picogram/nanogram DNA molecules, and is realized by optimizing specific nucleotide sequence primers and DNA probes and improving a bisulfite treatment method of plasma free DNA.
Description
Technical Field
The invention belongs to the technical field of biotechnology and DNA detection, and particularly relates to a DNA methylation qPCR kit for liver cancer detection and a using method thereof, in particular to a kit for determining the methylation state of one or more target gene targets (such as SEPT9) by means of methylation qPCR of plasma free DNA for liver cancer detection or screening and a using method thereof.
Background
Primary liver cancer is mainly hepatocellular carcinoma. Liver cancer is the fourth leading cause of cancer-related death worldwide, ranking the sixth in patient morbidity. The disease is hidden, has no specific clinical manifestation, is usually found to progress to the later stage, and misses the best treatment opportunity, so the prognosis is poor. The earlier the liver cancer is found, the more possible the liver cancer is cured, but the early liver cancer is generally asymptomatic, has no physical sign and is not easy to be found. The diagnosis and follow-up of the high risk group of liver cancer in clinic mainly depend on imaging and the detection of tumor marker AFP. However, the detection of imaging (B-ultrasound, CT, MRI, etc.) and CEA, AFP, etc. serum markers has certain limitations in the diagnosis of early liver cancer, the former has high equipment cost, strong requirements on operation technology and high detection cost; the latter is still insufficient for clinical detection due to its low sensitivity and specificity. Furthermore, the tissue biopsy as a standard for liver cancer diagnosis is highly invasive and is not suitable for screening early cancer. In addition, the traumatic detection mode brings great physiological pain to patients, so that the patients are not easy to accept. Therefore, more accurate and personalized means are urgently needed in the aspect of early diagnosis of liver cancer.
Liquid biopsy is a non-invasive method of replacing traditional tissue biopsy with a test molecular biomarker, and the biological sample (e.g., DNA) for analysis may be obtained from blood, urine, saliva, sputum, or tissue samples. Compared to traditional cancer diagnostic methods, liquid biopsy has the following advantages:
1. the liquid biopsy sample can be obtained from urine, saliva and pleural effusion;
2. less invasive than traditional tumor biopsy;
3. the ability to sample all possible cancer cells, rather than being limited to only a certain portion of the tumor biopsy;
4. facilitating early detection of cancer;
5. can be used to monitor tumor dynamics (pre-treatment, inter-treatment and post-treatment);
6. has the potential of being used as an ideal target identification therapeutic factor.
The DNA released into the blood by necrotic or apoptotic cells is called circulating free DNA (cfDNA), whereas in the blood of tumor patients a part of the cfDNA comes from dead tumor cells, which is defined as circulating tumor DNA (ctDNA), which is mainly composed of single-stranded or double-stranded DNA and a mixture of single-stranded and double-stranded DNA, exists in both forms of DNA protein complexes or free DNA, which is mostly a DNA fragment of about 134-144bp and carries certain tumor characteristics (including abnormal methylation, mutation, absence, insertion, copy number, etc.). Earlier cancers (meaning smaller tumor size and fewer cancer cells), release less free circulating tumor ctDNA, while their concentration is much less than free cfDNA; when tumor burden increases, ctDNA levels in patients increase. ctDNA is by far the best material for obtaining diagnostic, prognostic, and predictive information of molecular tumor-related changes in the blood of cancer patients.
DNA methylation changes are one of the earliest molecular changes in cancer progression and are tissue specific, and the high methylation levels of tumor suppressor genes have been identified as important molecular mechanisms that suppress gene expression and promote transformation of precancerous diseased tissue into malignant tumor cells. Among them, SEPT9 gene, human Septin9 gene, is located in AC068594 of chromosome 17q25, is a member of S-signen gene family, and is related to various cell functions of membrane vesicle transport to cytokinesis. Research shows that whether the promoter region of SEPT9 gene in ctDNA of a cirrhosis patient is methylated or not, whether the patient is diagnosed to develop liver cancer from cirrhosis or not is diagnosed, and SEPT9 can be used as an effective epigenetic marker and an individualized diagnosis label of hepatocellular carcinoma. Therefore, analysis of the methylation status of SEPT9 gene can be used to diagnose the status of liver cancer.
Methylation analysis of circulating tumor dna (ctDNA) can provide a viable option for cancer diagnosis, but the major challenge of clinical use of ctDNA to aid cancer diagnosis and therapy selection remains in how to develop highly sensitive detection methods to distinguish weak ctDNA signals at high cfDNA background levels, especially for early diagnosis where ctDNA concentrations may be on the order of pg/mL, with higher requirements on sensitivity and selectivity of the detection method. However, the current detection kit is limited by quantitative PCR methods such as poor DNA extraction method, poor bisulfite treatment capacity, and poor design of primers or probes for detecting methylated DNA, and thus the ctDNA methylated gene cannot be well utilized for early screening and auxiliary diagnosis of liver cancer. Therefore, there is an urgent need for more sensitive and accurate methods for detecting DNA methylation levels or states to aid in liver cancer diagnostic assays.
Disclosure of Invention
The invention aims to provide a blood monogene detection primer probe combination kit for early diagnosis, detection or screening of liver cancer, so as to overcome the defects of low specificity and sensitivity of a detection method in the prior art.
The first aspect of the invention provides a kit for detecting methylation of human SEPT9 gene, which is characterized by comprising specific primers and probes for detecting or measuring the methylation state or level of SEPT9 gene in DNA of a test sample.
In some embodiments of the invention, specific primers and probes are included to detect or measure the methylation status or level of the ACTB gene in the test sample DNA.
In some embodiments of the invention, the specific primers and probes included for detecting the methylation status of SEPT9 gene are selected from any one of the following three specific primer and probe combinations: 1-2 specific primers and 3 probes, 4-5 specific primers and 6 probes, 7-8 specific primers and 9 probes.
In some embodiments of the invention, specific primers SEQ ID NO 10-11 and probe SEQ ID NO 12 are included for detecting methylation status of the ACTB gene.
In some embodiments of the invention, the specific primers SEQ ID NO. 1, 2, 4, 5, 7, 8, 10, 11 are phosphorothioate modified and hybridize under stringent conditions to a region of the target gene that is methylated or unmethylated.
In some embodiments of the invention, the probes SEQ ID NO 3, 6, 9, 12 are designed based on TaqMan (TM) and hybridize under stringent conditions to a region of a target gene, which is methylated or unmethylated.
In some embodiments of the invention, the SEPT9 gene probe SEQ ID NO 3, 6, 9 nucleotide sequence is labeled ABY at the 5 'end and QSY at the 3' end.
In some embodiments of the invention, the ACTB gene probe SEQ ID NO 12 is labeled 5 'with VIC and 3' with MGBNFQ.
In some embodiments of the invention, the specific primers SEQ ID NO. 1, 2, 4, 5, 7, 8, 10, 11 and the probes SEQ ID NO. 3, 6, 9, 12 are 10-50nt in length.
In some embodiments of the present invention, the kit further comprises the following components: dNTP mixed solution, MgCl2Solution, DNA polymerase, PCR reaction buffer solution and PCR deionized water.
In some embodiments of the present invention, the kit further comprises the following components: a plasma free DNA extraction reagent and a plasma free DNA methylation conversion reagent.
In some embodiments of the invention, the plasma free DNA methylation conversion reagent is bisulfite.
In some embodiments of the invention, the test sample DNA is whole genome, cell-free DNA, or circulating tumor DNA.
In some embodiments of the invention, specific primer and probe sequences are shown in table 1 below:
TABLE 1 specific primers and probes contained in the kit of the invention
A second aspect of the present invention provides a method for using the kit of the first aspect, comprising the steps of:
(1) extracting plasma free DNA;
(2) performing methylation conversion on the extracted plasma free DNA and purifying, wherein bisulfite is preferably used as a reagent for performing methylation conversion on the plasma free DNA;
(3) carrying out fluorescent quantitative PCR amplification on the free DNA of the plasma subjected to methylation conversion and purification, setting a Ct threshold value in a linear amplification interval after the PCR reaction is finished, and determining that the amplification with the Ct value less than 40 is positive;
(4) and (4) judging a result: when the internal reference site ACTB detection result is positive, at least two of the three repeated amplifications of SEPT9 target point are positive, the result of the sample is determined to be positive.
In some embodiments of the invention, in step (3), PCR amplification of each template is performed in triplicate.
In some embodiments of the present invention, in the step (3), 10. mu.L of each reaction system comprises (0.5-1.5) xPCR buffer, 200-400 μm dNTPs, and 3-6mM MgCl21-3U AmpliTaq Gold DNA polymerase.
In some embodiments of the invention, the primers for the target regions of SEPT9 and SEPT9 gene in step (3) are 300nM and 500nM each.
In some embodiments of the invention, the probes for the target region of SEPT9 gene in step (3) are each 200-300 nM.
In some embodiments of the invention, in step (3), the primer for the target of the ACTB gene region is 150-250nM and the probe for the target of the ACTB gene region is 50-150 nM.
In some embodiments of the present invention, in the step (3), the PCR amplification conditions are: pre-denaturation at 90-100 deg.C for 8-12 min; denaturation at 90-100 deg.C for 10-20s, annealing at 60-70 deg.C and extension for 60-70s, and 40-50 cycles.
In some embodiments of the present invention, the step (3) is performed by any one of the following methods: methylation specific quantitative PCR, real-time methylation specific PCR, PCR using methylated DNA specific binding proteins.
The third aspect of the invention provides the use of specific primers and probes for detecting or measuring the methylation state or level of SEPT9 gene in test sample DNA in the preparation of kits;
preferably, the specific primers and probes for detecting or measuring the methylation state or level of SEPT9 gene in the test sample DNA are selected from any one of the following three specific primer and probe combinations: 1-2 specific primers and 3 probes, 4-5 specific primers and 6 probes, 7-8 specific primers and 9 probes.
The invention has the beneficial effects that:
through experimental tests, the kit can obviously improve the positive detection rate and specificity (real negative rate) of early liver cancer, can improve the detection sensitivity of the biomarker to picogram/nanogram DNA molecules, and is realized by optimizing specific nucleotide sequence primers and DNA probes and improving a bisulfite treatment method of plasma free DNA.
Drawings
FIG. 1: methylation qPCR amplification profile of blood samples from healthy persons.
FIG. 2: a liver cancer methylation qPCR amplification curve chart of a blood sample of a liver cancer patient.
FIG. 3: the results of the diagnostic value analysis (ROC curve) of liver cancer using the kit of examples 1 and 2 of the present invention are shown.
Detailed Description
The present invention will be described in detail and with reference to specific examples thereof, which are set forth to illustrate, but are not to be construed as the invention.
To make those skilled in the art understand the features and effects of the present invention, the terms and words used in the specification and claims are generally described and defined below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention, in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Definition of
The terms "patient," "individual," or "subject" are used interchangeably herein and may refer to a mammal, particularly a human. The subject may have mild, moderate or severe disease. The patient may be untreated, susceptible to treatment, or refractory. The patient may be an individual in need of treatment or diagnosis based on a particular symptom or family history.
The terms "sample," "patient sample," "biological sample," and the like include various sample types obtained from a patient, individual, or subject, and can be used for diagnostic or monitoring assays. The patient sample may be obtained from a healthy subject, a diseased patient, or a patient with symptoms associated with cervical cancer. Furthermore, the sample obtained from the patient may be segmented and only a portion may be used for diagnosis. In addition, the sample or a portion thereof may be stored under conditions that maintain the sample for later analysis. Specifically included within this definition are blood and other liquid samples of biological origin (including but not limited to peripheral blood, serum, plasma, urine, saliva, sputum, stool, and synovial fluid), solid tissue samples (such as biopsy specimens or tissue cultures or cells derived therefrom and progeny thereof). The definition also includes samples that are manipulated in any manner after being obtained, such as by centrifugation, filtration, precipitation, dialysis, chromatography, reagent treatment, washing, or enrichment for certain cell populations. These terms also include clinical samples, cultured cells, cell supernatants, tissue samples, organs, and the like. The sample may also comprise freshly frozen and/or formalin fixed paraffin embedded tissue blocks, such as blocks prepared by clinical or pathological biopsy, prepared for pathological analysis or by immunohistochemistry studies.
The terms "measuring," "determining," "detecting," or "examining" are used interchangeably throughout and may refer to a method that includes obtaining a patient sample and/or detecting a biomarker methylation status or level in a patient sample. In one embodiment, these terms refer to obtaining a patient sample and detecting the methylation state or level of one or more biomarkers in the sample. In another embodiment, the terms "measuring", "determining" or "detecting" refer to detecting the methylation status or level of one or more biomarkers in a patient sample. Measurement can be accomplished by methods known in the art and further described herein, including but not limited to methylation specific quantitative polymerase chain reaction (qPCR).
The term "methylation" refers to methylation of cytosine at the C5 or N4 position of cytosine, the N6 position of adenine, or other types of nucleic acid methylation. The in vitro amplified DNA is unmethylated because the in vitro DNA amplification method does not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA whose original template was unmethylated or methylated, respectively.
The term "CpG island" refers to a contiguous region of genomic DNA having a high density of CpG.
The term "methylation state" or "methylation level" refers to the presence, absence, and/or amount of methylation at a particular nucleotide or nucleotide in a portion of DNA.
It should be understood that wherever the language "comprising" is used to describe an embodiment, other similar embodiments described in "consisting of …" and/or "consisting essentially of …" are also provided.
Example 1: qPCR detection of liver cancer methylation on healthy human cfDNA sample by using kit
(I) test materials
1.10 parts of healthy human plasma;
QIAamp circulatory system nucleic acid extraction kit (purchased from Qiagen, Inc.);
EZ DNA Methylation-Lightning kit (available from Zymo Research Co.);
4. the kit (wherein the kit comprises a primer and probe combination 1 for detecting a target SEPT9, and an ACTB amplification primer and probe);
5.AmpliTaq GoldTMDNA polymerase and buffer reagents (available from Thermo Fisher).
(II) Experimental method
1. Plasma free DNA extraction
cfDNA was extracted from 10 healthy human plasma samples using QIAamp circulatory system nucleic acid extraction kit, the steps being performed according to the kit instructions.
2. Free DNA methylation conversion
Extracted 5 plasma cfDNA was bisulfite-treated and then purified using EZ DNA Methylation-Lightning kit from Zymo Research.
qPCR amplification
The bisulfite-treated plasma cfDNA was subjected to qPCR amplification using a Thermo Fisher Quant Studio 5 instrument, eachPCR amplification of the template was done in triplicate. 10 μ L of each reaction system containing 1xPCR reaction buffer, 400 μm dNTPs, 4mM MgCl22U AmpliTaq Gold DNA polymerase; 800nM each of the primers for the target of the SEPT9 gene region (only one pair of primers was selected for each target), 500nM each of the probes for the target of the SEPT9 gene region (one probe was selected for each target corresponding to the primer selected); primer 200nM of ACTB gene region target, probe 100nM of ACTB gene region target; 50nM ROX dye.
The PCR amplification conditions were: pre-denaturation at 95 ℃ for 10 min;
denaturation at 95 ℃ for 15s, annealing and extension at 65 ℃ for 30s, 45 cycles;
after the PCR reaction was completed, the Ct threshold was set in the linear amplification region (in this experiment,. DELTA.Rn was set to 0.1), and amplification with a Ct value of less than 40 was considered positive.
(III) results of the experiment
The criteria for the result is that when the internal reference site (ACTB) test is positive, at least two of the three duplicate amplifications at SEPT9 test site/target are positive and the sample is determined to be positive.
Table 2 below shows the results of 10 healthy human plasma samples determined using the present invention (fig. 1). As can be seen from Table 2, the detection specificity of this protocol is very high.
TABLE 2 results of the detection of blood samples of healthy persons using the kit of the present invention
Example 2: qPCR (quantitative polymerase chain reaction) detection experimental material for liver cancer methylation of ctDNA (deoxyribonucleic acid) sample of liver cancer patient by using kit
1.10 parts of blood plasma of a liver cancer patient;
QIAamp circulatory system nucleic acid extraction kit (purchased from Qiagen, Inc.);
EZ DNA Methylation-Lightning kit (available from Zymo Research Co.);
4. the kit (wherein the kit comprises a primer and probe combination 1 for detecting a target SEPT9, and an ACTB amplification primer and probe);
AmpliTaq GoldtMDNA polymerase and buffer reagent (available from Thermo Fisher Co.).
(II) Experimental method
1. Plasma free DNA extraction
ctDNA was extracted from 10 plasma samples of liver cancer patients using QIAamp circulatory system nucleic acid extraction kit, and the procedure was performed according to the kit instructions.
2. Free DNA methylation conversion
10 portions of the extracted plasma ctDNA were bisulfite-treated and then purified using EZ DNA Methylation-Lightning kit from Zymo Research.
qPCR amplification
The bisulfite treated plasma ctDNA was subjected to qPCR amplification using a Thermo Fisher Quant Studio 5 instrument with three replicates of PCR amplification per template. 10 μ L of each reaction system containing 1xPCR reaction buffer, 400 μm dNTPs, 4mM MgCl22U AmpliTaq Gold DNA polymerase; 800nM each of the primers for the target of the SEPT9 gene region (only one pair of primers was selected for each target), 500nM each of the probes for the target of the SEPT9 gene region (one probe was selected for each target corresponding to the primer selected); primer 200nM of ACTB gene region target, probe 100nM of ACTB gene region target; 50nM ROX dye.
The PCR amplification conditions were: pre-denaturation at 95 ℃ for 10 min;
denaturation at 95 ℃ for 15s, annealing and extension at 65 ℃ for 30s, 45 cycles;
after the PCR reaction was completed, the Ct threshold was set in the linear amplification region (in this experiment,. DELTA.Rn was set to 0.1), and amplification with a Ct value of less than 40 was considered positive.
(III) results of the experiment
The criteria for the result is that when the internal reference site (ACTB) test is positive, at least two of the three duplicate amplifications at SEPT9 test site/target are positive and the sample is determined to be positive.
Table 3 below shows the results of plasma samples of 10 liver cancer patients measured using the present invention (fig. 2). As can be seen from Table 3, the positive detection rate of this protocol was very high.
TABLE 3 test results of blood samples from patients with liver cancer using the kit of the present invention
By combining example 1 and example 2, it can be seen that the kit of the present invention for screening liver cancer has a positive detection rate of 80%, a specificity (true negative rate) of 90%, and a diagnostic value analysis (ROC curve) of 0.85 (fig. 3).
Different from example 1, the detection of the sample of the non-liver cancer control population and the blood sample of the liver cancer patient by using the primer and probe combination 2 containing the detection target SEPT9 and the kit of the primer and probe combination 2 containing the detection target SEPT9 respectively achieves similar results to example 1 and example 2.
While the preferred embodiments and examples of the present invention have been described in detail, the present invention is not limited to the embodiments and examples, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
Sequence listing
<120> kit for detecting human SEPT9 gene methylation and use method and application thereof
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 1
tgtcgggatt tggagttttc 20
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence ()
<400> 2
ctcatttcaa cacaaccttt cc 22
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence ()
<400> 3
ttcggagagg tttttaggtt ga 22
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence ()
<400> 4
<210> 5
<211> 19
<212> DNA
<213> Artificial sequence ()
<400> 5
ccaaacccac ccgcaaaat 19
<210> 6
<211> 19
<212> DNA
<213> Artificial sequence ()
<400> 6
tgttgggttg gttgttgcg 19
<210> 7
<211> 19
<212> DNA
<213> Artificial sequence ()
<400> 7
ggttgaggtc gcgtttttc 19
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 8
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 9
<210> 10
<211> 26
<212> DNA
<213> Artificial sequence ()
<400> 10
tagggagtat ataggttggg gaagtt 26
<210> 11
<211> 27
<212> DNA
<213> Artificial sequence ()
<400> 11
aacacacaat aacaaacaca aattcac 27
<210> 12
<211> 25
<212> DNA
<213> Artificial sequence ()
<400> 12
tggtgatgga ggaggtttag gcagt 25
Claims (10)
1. A kit for detecting human SEPT9 gene methylation, which is characterized by comprising specific primers and probes for detecting or measuring the methylation state or level of SEPT9 gene in test sample DNA.
2. The kit of claim 1, further comprising specific primers and probes for detecting or measuring the methylation state or level of the ACTB gene in the test sample DNA.
3. The kit according to claim 1 or 2, characterized in that the specific primers and probes for detecting the methylation state of SEPT9 gene are selected from any one of the following three specific primer and probe combinations: 1-2 specific primers and 3 probes, 4-5 specific primers and 6 probes, 7-8 specific primers and 9 probes;
and/or, comprises specific primers SEQ ID NO 10-11 and a probe SEQ ID NO 12 for detecting the methylation state of the ACTB gene.
4. The kit of any one of claims 1 to 3, wherein the specific primers SEQ ID NO 1, 2, 4, 5, 7, 8, 10, 11 are phosphorothioate-modified and hybridize under stringent conditions to a region of a target gene that is methylated or unmethylated;
and/or the probes SEQ ID NO 3, 6, 9, 12 are designed based on TaqMan (TM) and hybridize under stringent conditions to a region of a target gene, which is methylated or unmethylated.
5. The kit of any one of claims 1 to 4, wherein the SEPT9 gene probe SEQ ID NO 3, 6, 9 has a nucleotide sequence with a 5 'end labeled with Cy5 and a 3' end labeled with QSY;
and/or, the ACTB gene probe SEQ ID NO 12 nucleotide sequence is marked with VIC at the 5 'end and MGBNFQ at the 3' end.
6. The kit according to any one of claims 1 to 5, wherein the specific primers SEQ ID NO 1, 2, 4, 5, 7, 8, 10, 11 and the probes SEQ ID NO 3, 6, 9, 12 are 10 to 50nt in length.
7. The kit according to any one of claims 1 to 6, further comprising the following components: dNTP mixed solution, MgCl2Solution, DNA polymerase, PCR reaction buffer solution and PCR deionized water;
and/or, the kit also comprises the following components: a plasma free DNA extraction reagent and a plasma free DNA methylation conversion reagent;
and/or, the plasma free DNA methylation conversion reagent is bisulfite;
and/or, the test sample DNA is whole genome, cell-free DNA, or circulating tumor DNA.
8. A method of using the kit according to any one of claims 1 to 7, comprising the steps of:
(1) extracting plasma free DNA;
(2) performing methylation conversion on the extracted plasma free DNA and purifying, wherein bisulfite is preferably used as a reagent for performing methylation conversion on the plasma free DNA;
(3) carrying out fluorescent quantitative PCR amplification on the free DNA of the plasma subjected to methylation conversion and purification, setting a Ct threshold value in a linear amplification interval after the PCR reaction is finished, and determining that the amplification with the Ct value less than 40 is positive;
(4) and (4) judging a result: when the detection result of the internal reference site ACTB is positive, determining that the result of the target point is positive if at least two of the three repeated amplifications of the SEPT9 target point are positive;
preferably, in the step (3), the PCR amplification of each template is performed in three repetitions;
and/or 10. mu.L of each reaction system containing (0.5-1.5. mu.L) 1xPCR reaction buffer, 200-400. mu.m dNTPs, 3-6mM MgCl21-3U AmpliTaq Gold DNA polymerase;
and/or, the primers of the SEPT9 gene region target point are respectively 300-500 nM;
and/or, probes of the SEPT9 gene region target point are respectively 200-300 nM;
and/or, primer 150-250nM of ACTB gene region target, probe 50-150nM of ACTB gene region target;
and/or, the PCR amplification conditions are as follows: pre-denaturation at 90-100 deg.C for 8-12 min; denaturation at 90-100 deg.C for 10-20s, annealing at 60-70 deg.C and extension for 60-70s, and 40-50 cycles.
9. The method according to claim 8, wherein the step (3) is any one of the following methods: methylation specific quantitative PCR, real-time methylation specific PCR, PCR using methylated DNA specific binding proteins.
10. The application of specific primers and probes for detecting or measuring the methylation state or level of SEPT9 gene in the DNA of a test sample in the preparation of a kit;
preferably, the specific primers and probes for detecting or measuring the methylation state or level of SEPT9 gene in the test sample DNA are selected from any one of the following three specific primer and probe combinations: 1-2 specific primers and 3 probes, 4-5 specific primers and 6 probes, 7-8 specific primers and 9 probes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011414930.3A CN112322743A (en) | 2020-12-04 | 2020-12-04 | Kit for detecting human SEPT9 gene methylation and use method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011414930.3A CN112322743A (en) | 2020-12-04 | 2020-12-04 | Kit for detecting human SEPT9 gene methylation and use method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112322743A true CN112322743A (en) | 2021-02-05 |
Family
ID=74302533
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011414930.3A Pending CN112322743A (en) | 2020-12-04 | 2020-12-04 | Kit for detecting human SEPT9 gene methylation and use method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112322743A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114574584A (en) * | 2021-11-30 | 2022-06-03 | 江苏鹍远生物技术有限公司 | Tumor detection marker and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222267A (en) * | 2016-08-01 | 2016-12-14 | 博尔诚（北京）科技有限公司 | Compositions of detection hepatocarcinoma and application thereof |
| CN109112216A (en) * | 2018-07-30 | 2019-01-01 | 深圳市新合生物医疗科技有限公司 | The kit and method of triple qPCR detection DNA methylations |
| CN111560435A (en) * | 2020-05-20 | 2020-08-21 | 深圳市新合生物医疗科技有限公司 | DNA methylation kit for colorectal cancer detection, and use method and application thereof |
-
2020
- 2020-12-04 CN CN202011414930.3A patent/CN112322743A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222267A (en) * | 2016-08-01 | 2016-12-14 | 博尔诚（北京）科技有限公司 | Compositions of detection hepatocarcinoma and application thereof |
| CN109112216A (en) * | 2018-07-30 | 2019-01-01 | 深圳市新合生物医疗科技有限公司 | The kit and method of triple qPCR detection DNA methylations |
| CN111560435A (en) * | 2020-05-20 | 2020-08-21 | 深圳市新合生物医疗科技有限公司 | DNA methylation kit for colorectal cancer detection, and use method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| YURIKA KOTOH ET AL.: "Novel Liquid Biopsy Test Based on a Sensitive Methylated SEPT9 Assay for Diagnosing Hepatocellular Carcinoma", YURIKA KOTOH ET AL. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114574584A (en) * | 2021-11-30 | 2022-06-03 | 江苏鹍远生物技术有限公司 | Tumor detection marker and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109825586B (en) | DNA methylation qPCR kit for lung cancer detection and use method | |
| CN109112216B (en) | Triple qPCR (quantitative polymerase chain reaction) detection kit and method for DNA methylation | |
| CN110387421A (en) | DNA methylation qPCR kit and application method for lung cancer detection | |
| CN111560435A (en) | DNA methylation kit for colorectal cancer detection, and use method and application thereof | |
| CN109504780B (en) | DNA methylation qPCR kit for lung cancer detection and use method thereof | |
| EP3800273A1 (en) | Use of detection reagent for detecting methylation of genes associated with colorectal cancer, and kit | |
| CN110964823A (en) | DNA methylation kit for colorectal cancer detection and detection method | |
| EP2210942B1 (en) | Gene associated with liver cancer, and method for determination of the risk of acquiring liver cancer | |
| CN111549135A (en) | DNA methylation qPCR kit for cervical cancer detection, and use method and application thereof | |
| CN112646888B (en) | Kit for detecting mammary tumor specific methylation | |
| CN113308544B (en) | Reagent for DNA methylation detection and esophageal cancer detection kit | |
| CN113355415B (en) | Detection reagent and kit for diagnosis or auxiliary diagnosis of esophageal cancer | |
| CN108220428B (en) | Composition for detecting gastric cancer, kit and application thereof | |
| CN111500731A (en) | Kit for early diagnosis, detection or screening of thyroid cancer, and use method and application thereof | |
| CN111808962A (en) | Kit for cervical cancer detection and use method | |
| CN109055555B (en) | Lung cancer early stage metastasis diagnosis marker and kit and application thereof | |
| CN111793690A (en) | DNA methylation qPCR kit for cervical cancer detection and use method thereof | |
| CN113355414A (en) | Esophageal cancer detection kit and application thereof | |
| CN112322743A (en) | Kit for detecting human SEPT9 gene methylation and use method and application thereof | |
| CA3181473A1 (en) | Tumor detection reagent and kit | |
| CN111826446A (en) | Primer, probe and kit for early screening and auxiliary diagnosis of bladder cancer | |
| CN111254199A (en) | Lung cancer related KEAP1 gene methylation detection kit | |
| CN107326092B (en) | Application of colorectal cancer-related miRNA as biomarker and colorectal cancer detection kit | |
| US7217515B2 (en) | HURP gene as a molecular marker for bladder cancer | |
| CN112391478B (en) | Application of exosome mRNA in diagnosis of breast diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |